Hu sheep lambing number prediction method based on EPAS1 gene variation and application of Hu sheep lambing number prediction method

Abstract

The invention discloses application of an SNP (Single Nucleotide Polymorphism) marker influencing the lambing number of sheep in an EPAS1 gene. The marker is located on an EPAS1 gene of a sheep chromosome 3, the specific SNP marker site is T / C base mutation at the 461bp position of SEQ ID NO: 1 in a sequence table, and the lambing number of TT genotype sheep at the site is remarkably higher than that of CC genotype individuals. The Hu sheep is used as a research object, PCR is used for amplifying SNP variation of the 17th exon of the sheep EPAS1 gene, the influence of the specific genotype of the variation site on the lambing number of the Hu sheep is analyzed, breeding of Hu sheep individuals is performed according to the influence, the method can be used for increasing the lambing number of the Hu sheep, and marker resources are provided for marker-assisted selective breeding of the lambing number character of the sheep.

Description

Technical Field

[0001] The invention belongs to the technical field of animal molecular breeding, and relates to the application of SNP markers related to the trait of litter size of Hu sheep in Hu sheep breeding. Background Art

[0002] The number of lambs born is a key indicator of the reproductive capacity of domestic animals and directly affects the breeding benefits. Hu sheep is a fine local sheep breed in China with a high reproduction rate and strong adaptability. In the plateau environment, the low oxygen environment affects the physiological state of the ewes, especially during pregnancy, when the ewes need a higher oxygen supply to maintain the normal development of themselves and the fetus. Therefore, the low oxygen environment of the plateau may affect the number of lambs born by Hu sheep, which poses new challenges and demands for maintaining the high reproductive performance of Hu sheep.

[0003] EPAS1, also known as hypoxia-inducible factor 2α (HIF-2α), is an important member of the hypoxia-inducible factor family and can sense and respond to changes in intracellular oxygen concentration. Under normal oxygen levels, HIF-2α is ubiquitinated and degraded, while under hypoxic conditions, HIF-2α is in a stable state, enters the nucleus and binds to HIF-1β, activating the expression of multiple hypoxia response-related genes. EPAS1 plays a vital role in erythropoiesis, iron metabolism, and angiogenesis. It directly promotes the production of red blood cells by regulating the expression of erythropoietin (EPO), thereby improving the oxygen carrying capacity of blood in a hypoxic environment. EPAS1 can also upregulate the expression of divalent metal ion transporters (DMT1) and cytochrome b (DcytB) in the intestine, enhance iron absorption and utilization, and support erythropoiesis. EPAS1 regulates the expression of vascular endothelial growth factor (VEGF), promotes angiogenesis, increases the number and permeability of placental blood vessels, provides sufficient oxygen and nutrients to the fetus, and is beneficial to the development of pregnant ewes and their fetuses. Therefore, the EPAS1 gene may be involved in regulating the reproductive performance of sheep. The present invention found SNP markers in the EPASI gene that are related to the number of lambs born in Hu sheep, indicating that the EPAS1 gene is involved in the reproduction of Hu sheep under hypoxic conditions, and provides genetic marker resources for marker-assisted selection of the number of lambs born in Hu sheep. Summary of the invention

[0004] The invention provides a method for solving the technical problem of the existing sheep breeding lamb number.

[0005] According to an embodiment of the present invention, the SNP marker is located at the 461th base of the nucleotide sequence shown in SEQ ID NO: 1, and the number of lambs born by Hu sheep individuals with TT genotype at the SNP marker site is significantly higher than that of individuals with CC genotype. By detecting the above-mentioned SNP marker of Hu sheep, its number of lambs can be effectively predicted, so that the lambing performance of Hu sheep individuals can be evaluated according to the genotype of the SNP marker site. Therefore, the SNP marker of the present invention is closely related to the lambing quantity trait of Hu sheep, and can be effectively applied to the molecular marker-assisted breeding of Hu sheep, and then the sheep breeding materials can be selected according to the actual breeding needs, so as to accurately and efficiently screen out high-quality individuals with multiple lambs, and improve the efficiency and accuracy of breeding selection.

[0006] The present invention provides a primer pair for detecting the SNP marker of claim 1. According to an embodiment of the present invention, the primer pair has the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence table, and is used for detecting the SNP marker.

[0007] According to an embodiment of the present invention, the present invention provides a method for predicting the high lamb number of sheep. The primer pair, amplification program and amplification system of the present invention can effectively amplify the fragment where the SNP marker related to the lamb number trait of the tested Hu sheep is located. The detection of this SNP marker can be effectively achieved by sequencing, and the genotype of the SNP marker site of the tested Hu sheep can be determined, so as to effectively predict the lamb number of the tested Hu sheep. Specifically, the TT genotype of the SNP marker site can be used as an important criterion for judging the number of lambs produced by Hu sheep. In the Hu sheep breeding work, sheep with the genotype of the SNP marker site TT genotype can be retained for breeding, and sheep with the genotype of the SNP marker site CC and TC can be eliminated. Therefore, the present invention can be effectively applied to the molecular marker-assisted selection breeding of Hu sheep, so as to screen out Hu sheep individuals with a large number of lambs at low cost and high accuracy.

[0008] The present invention has the following beneficial effects: (1) The SNP markers provided by the present invention are significantly correlated with the number of lambs born by Hu sheep, and the number of lambs born by sheep with TT genotype is significantly higher than that of sheep with CC genotype. (2) By identifying the above-mentioned specific SNP markers, it can help breeders provide accurate basis for selecting high-lamb-producing individuals, thereby enhancing the scientificity and reliability of breeding. (3) The detection method provided by the present invention is easy to operate, and the genotype of the target SNP site can be accurately detected by using PCR amplification and conventional sequencing technology. This method is not only low-cost, but also suitable for large-scale screening and application, and has good prospects for promotion and application. Specific embodiments

[0009] The embodiments of the present invention are described in detail below, and the present invention is further described in detail in conjunction with the embodiments. The embodiments herein are only used to illustrate the present invention and are not to be construed as limiting the present invention.

[0010] 1. Experimental samples The experimental samples in this embodiment were obtained from Hubei Zhiqinghe Agriculture and Animal Husbandry Co., Ltd., and blood samples were collected from 84 adult Hu sheep ewes, whose feeding and management conditions and environmental conditions were consistent. Jugular vein blood (5 mL / goat) was collected using a disposable vacuum negative pressure blood collection tube (EDTA-K2 anticoagulation), and the samples were stored at -20°C.

[0011] 2. Genomic DNA Extraction The genomic DNA from the blood samples of Hu sheep was extracted using the phenol-chloroform method. The extracted DNA was tested for quality using 1.0% agarose gel to ensure that the integrity and concentration of the DNA sample were suitable for subsequent experiments.

[0012] 3. Primer Design According to the sequence of sheep EPAS1 gene (Ensembl database gene sequence number: ENSOARG00000005722), a pair of specific primers SEQ ID NO: 2 and SEQ ID NO: 3 were designed using the NCBI online primer design tool. The primers were synthesized by Wuhan Jinkairui Biotechnology Co., Ltd. The specific primers were used to amplify a DNA sequence where the 17th exon of the EPAS1 gene is located. The amplified product was 939 bp, and the DNA sequence is shown in SEQ ID NO: 1 in the sequence table.

[0013] 4. PCR amplification and sequencing of sheep EPAS1 gene target sequence (1) PCR amplification system (25 μL): DNA 1 μL, primers SEQ ID NO: 2 and SEQ ID NO: 3 (10 μM) 1 μL each, 2×Rapid Taq Master Mix 12.5 μL (Beijing Quanshijin Biotechnology Co., Ltd.), ddH2O 9.5 μL. Amplification program: 95°C pre-denaturation for 5 min, 95°C denaturation for 40 s, 63°C annealing for 40 s, 72°C extension for 5 min, 35 cycles. (2) The PCR amplification product was sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. The sequencing results were read using the SeqMan tool in DNASTAR to determine the genotype of the individual at the 461 bp position of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence list.

[0014] 5. Association analysis between EPAS1 gene SNP markers and litter size in Hu sheep SAS8.1 software was used to analyze the association between genotype and litter size. The specific mixed linear model is as follows: Y ij =μ+G i +e ij where Y ij is the number of lambs born in the first litter; μ is the population mean; G j is the genotype effect; e ij is the random effect residual.

[0015] 6. Analysis of the significance of the difference in the number of lambs born among Hu sheep with different genotypes The results of the analysis of the number of lambs of different genotypes of Hu sheep are shown in Table 1. As can be seen from Table 1, there are three genotypes at this locus, and the difference in the number of lambs between different genotypes was compared by one-way analysis of variance. According to the analysis results, it was found that the number of lambs of Hu sheep with TT genotype was significantly higher than that of CC genotype (p<0.05), and the number of lambs of Hu sheep with TC genotype was no different from that of sheep with TT and CC genotypes (p>0.05). This shows that the TT genotype of this SNP marker locus can be used as an important criterion for judging the high number of lambs of Hu sheep. In the process of Hu sheep breeding, sheep with SNP marker TT genotype are selected and retained for breeding, and individuals with genotypes of TC and CC are eliminated, which can effectively improve the number of lambs of Hu sheep populations. Table 1. Correlation between different genotypes of EPAS1 gene SNP loci and the average number of lambs born in three litters of Hu sheep Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

Claims

1. A method for selecting sheep based on a molecular marker related to the lamb number trait of sheep, wherein the molecular marker is located in the 17th exon region of the sheep EPAS1 gene, and the specific nucleotide sequence is SEQ ID NO: 1, wherein the lamb number of sheep individuals with TT genotype at the 461 bp position of the sequence is significantly higher than that of sheep individuals with CC genotype; The method comprises the following steps: (1) Extracting sheep genomic DNA; (2) using two specific primers to perform PCR amplification to obtain a 939 bp amplification product, wherein the sequences of the two specific primers are SEQ ID NO: 2 and SEQ ID NO: 3; (3) sequencing the PCR amplification product to obtain a sequencing result; (4) determining the genotype of the individual sheep to be tested at the molecular marker according to the sequencing results; (5) selecting sheep individuals with the TT genotype of the above molecular markers for breeding; (6) The sheep are Hu sheep.

2. Use of the SNP marker described in claim 1 in screening Hu sheep with high lamb production, and selecting sheep individuals with the TT genotype of the molecular marker for breeding.

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